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Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161 and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here, we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or nuclear egress complex (NEC) interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that misregulated budding may not substantially impair virus replication. In addition, the R158A substitution caused relocalization of the NEC to intranuclear punctate structures and recruited lamin A/C to these structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. [ABSTRACT FROM AUTHOR]

Rhamnolipids (RLs) comprise a class of glycolipids produced by Pseudomonas aeruginosa under appropriate culture medium. They act as biosurfactants being composed by a hydrophilic head of either one (mono-RL) or two (di-RL) rhamnose moieties coupled to hydroxyaliphatic chains. It is well accepted that RLs present low biolitic activity as compared to other synthetic surfactants. However, their mechanisms of action in biological systems are not well defined yet. The interaction of RLs with lipid bilayers are here investigated to address how they impact on plasma membrane at molecular level. Our experimental approach was based on a deep analysis of optical microscopy data from giant unilamellar vesicles (GUVs) dispersed in aqueous solutions containing up to 0.5 mM of commercially available RLs (a mixture of mono-RL, 33–37 mol%, and di-RL, 63–67 mol%, cmc of 0.068 ± 0.005 mM). GUVs were made up of a single lipid POPC and a ternary system containing DOPC, sphingomyelin and cholesterol, which mimic lipid raft platforms. Our results demonstrate that RLs have a low partition in the lipid bilayer in respect to the total molecules in solution. We suppose that RLs insert in the outer leaflet with low propensity to flip-flop. In the case of POPC GUVs, the insertion of RL molecules in the outer leaflet impairs changes in spontaneous membrane curvature with incubation time. Then, small buds are formed that remain linked to the original membrane. No changes in membrane permeability have been detected. A remarkable result refers to the insertion of RLs in membranes containing liquid ordered ( L o ) - liquid disordered ( L d ) phase coexistence. The rate of interaction has been observed to be higher for L d phase than for L o phase (0.12 · 10 - 6 s−1 and 0.023 · 10 - 6 s−1 for L d and L o , respectively, at RL concentration of 0.5 mM). As a consequence, the preferential RL insertion in L d phase may also alter the membrane spontaneous curvature which, coupled to the change in the line tension associated to the domains boundary, conducted to L o domain protrusion. Even if it has been observed on a model system, such membrane remodelling might correlate to endocytic processes activated in cell membranes, regardless of the participation of specific proteins. Further, changes imposed by RLs in lipid rafts may affect the association of key proteins enrolled in cell signaling, which may perturb cell homeostasis. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A scaffolding NP system was proposed to extract lipid vesicles from the membrane. • Some design principles were revealed by Dissipative Particle Dynamics simulations. • The vesicle size varies depending on NP concentration and membrane surface tension. • Future experiments can be guided to fabricate NPs for use in single-cell analysis. Abstract Single-cell analysis is a new and rapidly expanding field, the goal of which is obtaining fresh information from individual cells to understand the regulatory mechanisms of cell development and diseases. Conventional approaches generally rely on the cell lysis which, however, is destructive to cells and against multiple sampling from the living cell. Here, we propose and design a scaffolding nanoparticle (NP) system that enables us to sample cytoplasmic contents without rupturing the cellular membrane, by mimicking the unusual features of clathrin. Our simulation results reveal the design principles, following which scaffolding NPs can extract lipid vesicles from plasma membranes, with both the pathway and the mechanism resembling the clathrin-mediated endocytosis, i.e. multiple NPs deposit at the membrane, assembling into cage-like structures to deform the membrane into a vesicle shape. As important design parameters, the interaction between different NPs should be properly stronger than that between each NP and the membrane to ensure the cage formation, and optimal NP concentration and the membrane surface tension are also requisite for extracting lipid vesicles. Our results provide useful guidelines for design of bio-inspired scaffolding NPs as an intelligent machine for practical use in but not limited to the single-cell analysis. [ABSTRACT FROM AUTHOR]

We study the distension-induced gradient capillarity in membrane bud formation. The budding process is assumed to be primarily driven by diffusion of transmembrane proteins and acting line tensions on the protein-concentrated interface. The proposed model, based on the Helfrich-type potential, is designed to accommodate inhomogeneous elastic responses of the membrane, non-uniform protein distributions over the membrane surface and, more importantly, the thickness distensions induced by bud formations in the membrane. The latter are employed via the augmented energy potential of bulk incompressibility in a weakened manner. By computing the variations of the proposed membrane energy potential, we obtained the corresponding equilibrium equation (membrane shape equation) describing the morphological transitions of the lipid membrane undergoing bud formation and the associated thickness distensions. The effects of lipid distension on the shape equation and the necessary adjustments to the accompanying boundary conditions are also derived in detail. The resulting shape equation is solved numerically for the parametric representation of the surface which has one-to-one-correspondence with the membrane surface under consideration. The proposed model successfully predicts the bud formation phenomenon on a flat lipid membrane and the associated thickness distensions of the membrane and demonstrates a smooth transition from one phase to the other (including necking domains). It is also found that the final deformed configuration is energetically favorable and therefore is stable. Finally, we show that the inhomogeneous thickness deformation on the membrane in response to transmembrane protein diffusion makes a significant contribution to the budding and necking processes of the membrane. [ABSTRACT FROM AUTHOR]

We study the formation of membrane budding in model lipid bilayers with the budding assumed to be driven by means of diffusion of trans-membrane proteins over a composite membrane surface. The theoretical model for the lipid membrane incorporates a modified Helfrich-type formulation as a special case. In addition, a spontaneous curvature is introduced into the model in order to accommodate the effect of the non-uniformly distributed proteins in the bending response of the membrane. Furthermore, we discuss the effects of line tension on the budding of the membrane, and the necessary adjustments to the boundary conditions. The resulting shape equation is solved numerically for the parametric representation of the surface, which has one to one correspondence to the membrane surface in consideration. Our numerical results successfully predict the vesicle formation phenomenon on a flat lipid membrane surface, since the present analysis is not restricted to the conventional Monge representation often adopted to the problems of this kind for the obvious computational simplicity, despite its limited capability to describe the deformed configuration of membranes. In addition, we show that line tension at the interface of the protein-concentrated domain makes a significant contribution to the bud formation of membranes. [ABSTRACT FROM AUTHOR]

During nuclear egress, herpesvirus capsids bud at the inner nuclear membrane forming perinuclear viral particles that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytoplasm. This unusual budding process is mediated by the nuclear egress complex (NEC) composed of two conserved viral proteins, UL31 and UL34. Earlier, we discovered that the herpesvirus nuclear egress complex (NEC) could bud synthetic membranes in vitro without the help of other proteins by forming a coat-like hexagonal scaffold inside the budding membrane. To understand the structural basis of NEC-mediated membrane budding, we determined the crystal structures of the NEC from two herpesviruses. The hexagonal lattice observed in the NEC crystals recapitulates the honeycomb coats within the budded vesicles. Perturbation of the oligomeric interfaces through mutagenesis blocks budding in vitro confirming that NEC oligomerization into a honeycomb lattice drives budding. The structure represents the first atomic-level view of an oligomeric array formed by a membrane-deforming protein, making possible the dissection of its unique budding mechanism and the design of inhibitors to block it. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria produce outer membrane vesicles (OMVs) that contain biologically active proteins and perform diverse biological processes. Unlike other secretion mechanisms, OMVs enable bacteria to secrete insoluble molecules in addition to and in complex with soluble material. OMVs allow enzymes to reach distant targets in a concentrated, protected, and targeted form. OMVs also play roles in bacterial survival: Their production is a bacterial stress response and important for nutrient acquisition, biofilm development, and pathogenesis. Key characteristics of OMV biogenesis include outward bulging of areas lacking membrane-peptidoglycan bonds, the capacity to upregulate vesicle production without also losing outer membrane integrity, enrichment or exclusion of certain proteins and lipids, and membrane fission without direct energy from ATP/GTP hydrolysis. Comparisons of similar budding mechanisms from diverse biological domains have provided new insight into evaluating mechanisms for outer membrane vesiculation. [ABSTRACT FROM AUTHOR]

The adhesion of lipid vesicles to a rigid flat surface is investigated. We examine the influence of the membrane spontaneous curvature, adhesion strength, and the reduced volume on the stability and shape transformations of adhered vesicles. The minimal strength of the adhesion necessary to stabilize the shapes of adhered vesicles belonging to different shape classes is determined. It is shown that the budding of an adhered vesicle may be induced by the change of the adhesion strength. The importance of the free vesicle shape for its susceptibility to adhesion is discussed. [ABSTRACT FROM AUTHOR]

Biological membranes are composed of isotropic and anisotropic curved nanodomains. Anisotropic membrane components, such as Bin/Amphiphysin/Rvs (BAR) superfamily protein domains, could trigger/facilitate the growth of membrane tubular protrusions, while isotropic curved nanodomains may induce undulated (necklace-like) membrane protrusions. We review the role of isotropic and anisotropic membrane nanodomains in stability of tubular and undulated membrane structures generated or stabilized by cyto- or membrane-skeleton. We also describe the theory of spontaneous self-assembly of isotropic curved membrane nanodomains and derive the critical concentration above which the spontaneous necklace-like membrane protrusion growth is favorable. We show that the actin cytoskeleton growth inside the vesicle or cell can change its equilibrium shape, induce higher degree of segregation of membrane nanodomains or even alter the average orientation angle of anisotropic nanodomains such as BAR domains. These effects may indicate whether the actin cytoskeleton role is only to stabilize membrane protrusions or to generate them by stretching the vesicle membrane. Furthermore, we demonstrate that by taking into account the in-plane orientational ordering of anisotropic membrane nanodomains, direct interactions between them and the extrinsic (deviatoric) curvature elasticity, it is possible to explain the experimentally observed stability of oblate (discocyte) shapes of red blood cells in a broad interval of cell reduced volume. Finally, we present results of numerical calculations and Monte-Carlo simulations which indicate that the active forces of membrane skeleton and cytoskeleton applied to plasma membrane may considerably influence cell shape and membrane budding. [ABSTRACT FROM AUTHOR]

Summary Cells convert excess energy into neutral lipids that are made in the endoplasmic reticulum (ER) bilayer. The lipids are then packaged into spherical or budded lipid droplets (LDs) covered by a phospholipid monolayer containing proteins. LDs play a key role in cellular energy metabolism and homeostasis. A key unanswered question in the life of LDs is how they bud off from the ER. Here, we tackle this question by studying the budding of artificial LDs from model membranes. We find that the bilayer phospholipid composition and surface tension are key parameters of LD budding. Phospholipids have differential LD budding aptitudes, and those inducing budding decrease the bilayer tension. We observe that decreasing tension favors the egress of neutral lipids from the bilayer and LD budding. In cells, budding conditions favor the formation of small LDs. Our discovery reveals the importance of altering ER physical chemistry for controlled cellular LD formation. [ABSTRACT FROM AUTHOR]